Methyl p-hydroxyphenyllactate (MeHPLA) is an endogenous ligand for nuclear type II sites derived from bioflavonoid and/or tyrosine metabolism. The binding of MeHPLA to this site (Kd~5 nM) correlates with the inhibition of normal and malignant cell proliferation. MeHPLA is found in all normal tissues. However, this compound is hydrolyzed to p-hydroxyphenyllactic acid (HPLA) by esterases in malignant cells. HPLA binds to type II sites with low affinity (Kd>200 nM) and does not inhibit cell proliferation. therefore, the deficiency of MeHPLA in tumors correlates with the loss of regulatory control. We proposed (ACS grant CH-469) to synthesize esterase stable enones and chalcones which mimic MeHPLA for assessment as antitumor agents. We developed four such compounds (MV-3, MV-12, MV-88 and RSV-101) which bind to type II sites with high affinity (Kd~1-10 nM) and inhibit cancer cell proliferation in vitro and in vivo (see progress report). Furthermore, lower doses of these enones and chalcones enhance the activity of classical anticancer drugs such as 5-Fluorouracil (FU). Although slight inhibition of mouse mammary tumor growth was observed with FU alone, combination therapy with MV-88+FU or RSV-101+FU appeared curative in 90% of these animals. We expect that these enones and chalcones may also """"""""synergize"""""""" with Cytoxan (CY; cyclophosphamide) and Methotrexate (MTX). The objectives of the proposed project are to modify MV-3, MV-12, MV-88 and RSV-101 for optimum delivery and to define treatment protocols for maximum antitumor activity (specific aim 1). The effectiveness of MV-3, MV-12, MV- 88 and RSV-101 alone and with various combinations of FU, MTX or CY on mammary tumor growth will be evaluated (specific aim 2). Binding interactions with nuclear type II sites and subsequent effects on mammary cancer cell proliferation in vitro (human breast cancer cells) and anti- tumor activity in vivo (mouse mammary tumors; human breast cancer cells grown in athymic nude mice) will be assessed (cell growth inhibition, cell cycle kinetics, DNA polymerase activity). [3H]MV-3 and [3H]MV-88 will be administered to tumor bearing animals and the radioactive components identified to determine the biologically active form of these compounds in malignant cells (specific aim 3). These data will facilitate the further design of specific compounds and treatment strategies (singular and combination therapy) for maximum therapeutic activity.
